The leukaemias are a heterogeneous group of neoplasms arising from the malignant transformation of heamatopoietic i.e. blood forming cells. Leukaemia can be broadly classified according to the cell type involved primarily (myeloid or lymphoid) and as acute or chronic depending on the natural history of the disease.
Acute lymphoblastic leukaemia (ALL) is the commonest type of leukaemia in childhood. It is a primarily a disease of children and young adults. It occurs in all races with a peak incidence in children between 3 to 5 years of age. Acute lymphoblastic leukaemia (ALL) is diagnosed in 2000 to 3000 new cases of childhood leukaemia in United States each year, whereas Acute myelogenous leukaemia (AML) is diagnosed in only 500 children and Chronic myeloid leukaemia (CML) is fewer than 100. About 40 million children in U.S.A. are affected under the group of 15 years, about 3/4 Th. of these have ALL. (Nelson essential of Paedratics, 1990 Ed Bchrman RE and Klregman R pp. 53).
The causes are not known, but environmental agents including irradiation, chemical carcinogens, cytogenetic abnormalities and retrovirus infections are known to play an important role in the etiology of leukaemia. For instance, individuals with occupational/accidental radiation exposure, patients receiving radiation therapy or survivors of the atomic bomb explosions in Japan have a predictable and dose related increased incidence of leukaemia.
Pediatric haematopoietic malignancies rank first in cancer incidence and mortality in children and are responsible for roughly 40% of childhood related death (Carp and McCaffrey J. Natl. Cancer. Inst. 86, 1196, 1994).
In fact, the diagnosis and the treatment of Acute lymphoblastic leukaemia (ALL) is one of the major success stories of modern clinical oncology. For oncologists, Acute lymphoblastic leukaemia (ALL) represents a major therapeutic success as remission can be achieved in nearly 65% of patients (Pui and Crist Curr. Opinion on Oncology 7, 36, 1995, Lancet 374, pp 1783, 1996). With the available chemotherapeutic treatment, most of the Acute lymphoblastic leukaemia (ALL) patients can lead a normal disease free life for approximately 3-5 years. However, relapse and eventual treatment failure invariably occurs in most cases receiving identical treatment and this area is a major challenge for leukaemia specialists (Pui and Crist N. Eng. J. Med. 332, 1618, 1995). The reason behind the occurrence of relapse can be well understood if we take into consideration the definition adopted by the clinicians to state whether the patient has attained complete remission or not. The clinicians state:
a. If the laeukemia blasts in the bone marrow of the patients is less than 5% and PA1 b. If no laeukemia blast is detected in the peripheral blood of patient, the patient is said to be in the state of apparently complete remission. PA1 1. Flow cytometric analysis can detect one laeukemia blast cell in a total cell population of 1000. So it is said to have a sensitivity of 0.1% or 10.sup.-3, which is inferior to available DNA based method (Huh and Andreef 8, 713, 1994, Meydan et al. Nature 379, pp 645, 1996). PA1 2. The flow cytometer used for Flow cytometric analysis is very costly (about U.S. $1500) and not available in clinics. PA1 3. It requires mandatory technical expertise. PA1 4. The method needs expensive chemicals e.g. several specific antibodies and fluorescence conjugated chemicals. PA1 (i) occurrence of false positives due to contamination of reaction mix with previously employed samples PA1 (ii) occurrence of false negatives results owing to degraded RNA or DNA or clonal evolution in approximately 20% of cases (Pui., 1995) PA1 (iii) not all laeukemia specific IgH and TCR gene rearrangements are amenable to initial amplification of PCR using universal primers PA1 (iv) a heterogeneous distribution of minimal residual disease (MRD) may result in sampling error since IgH and TCR gene rearrangements may be different in different individuals. PA1 (v) these methods are costly, lengthy, very sophisticated and requiring mandatory technical expertise. PA1 1. As stated by the title, the paper identifies O-acetylated sialic acid as a distinct marker for differentiation between several leukemia erythrocytes only and not specifically to Acute lymphoblastic leukaemia (ALL). Therefore, it deals with the identification of O acetylated glycoconjugate on erythrocytes only. PA1 2. The paper summarizes the result of a study of binding of AchatininH with erythrocytes of patients suffering from Acute lymphoblastic leukemia (ALL) in their acute phase only. PA1 3. In the above referred paper, very few patients (only five Acute lymphoblastic leukaemia (ALL) patients) have been included in the study and hence the result should not be considered as statistically significant. PA1 4. In the above referred paper, the method is hemagglutination assay with erythrocytes but not the lymphoproliferation assay with peripheral blood mononuclear cells. PA1 5. The study includes newly diagnosed Acute lymphoblastic leukaemia (ALL) patients who have not received any chemotherapeutic treatment. Thus, the preliminary work was only with the acute phase of the disease. This is nothing to do with the detection of minimal residual disease (MRD). PA1 6. the hemogglutination assay with erythrocytes has also been performed with Acute lymphoblastic leukaemia (ALL) patients at different stages of treatment, i.e., Phases A, B, C, D and E no agglutination could be observed. PA1 1. Detecting minimal residual disease--in lymphoid malignancies using the polymerase chain reaction. Drawbacks are a) It is specific for lymphoid malignancies i.e. chronic and acute lymphoblastic leukaemia and not just ALL and it is PCR based. PA1 2. BCL--3 gene sequences--used to detect cellular oncogenic C-myc translocation, leukaemia(s), and to monitor anti-neoplastic therapy. Drawbacks are a) It is not specific for acute lymphoblastic leukaemia buy any neoplastic transformation resulting due to C-myc oncogenic expression and is PCR based. Moreover detection of minimal residual disease (MRD) is not mentioned as per the method. PA1 3. DNA encoding human common acute lymphoblastic leukaemia antigen-used for obtaining pure protein for diagnosis and treatment of medial-conditions. Drawbacks are the following a) The method is PCR based and detection of MRD is not mentioned as per the method. PA1 (i) Peripheral blood mononuclear cells (PBMC) of Acute lymphoblastic leukaemia (ALL) patients, patients of other hematological disorders (AML, CML, NHL and Thalassemia) and normal donors were isolated by Ficoll Hypaque density centrifugation. PA1 (ii) PBMC (1.times.10.sup.5 cells/well) were cultured with different concentrations of Achatinin.sub.H (0.05-10 ug) in Medium A for 96 hours at 37.degree. C. with 5% CO.sub.2 and 95% air. PA1 (iii) The cultures were pulsed with .sup.3 H-Thymidine (1 uCi/well) for 18 hours. PA1 (iv) The cells were harvested and counts taken in Rack Beta LKB liquid scintillation counter. PA1 (v) For each patient and normal donor, the counts obtained were plotted in y axis against the Achatinin.sub.H dose plotted on x axis which ranged from 0.05-10 ug. PA1 (vi) From this plot, the dose of Achatinin.sub.H at which maximal incorporation of radioactivity is obtained was determined. This has been designated as the maximal lymphoproliferative dose of Achatinin.sub.H for that patient and normal donor. PA1 (i) Maximal proliferative dose of mitogen is very low and PA1 (ii) the density of the cell surface receptor to which the mitogen binds for mediating cellular proliferation is very high.
Therefore, the flaw lies in the definition itself since it states that leukaemic blast cells within a range of 0-5% may still be present within the patient when he is said to have attained the so called complete remission. At the time of diagnosis, the leukaemic cell mass is usually between 10.sup.11 -10.sup.12 cells and available chemotherapeutic agents produce a fractional cell kill capable of a 3 to 5 log kill resulting in the elimination of 99.99 to 99.999% of leukaemia cells. The remaining 0.01 to 0.001% leukaemic cells tantamounts to the persistence of 10.sup.8 to 10.sup.9 leukaemic blast cells respectively (Champlin and Golde Harrison Text book of Internal Medicine, 1552). What is important is that these persisting leukaemic blast cells are not detectable by standard morphology in bone marrow or peripheral blood. It is these cells that are responsible or relapse, if post induction chemotherapy fails to eradicate them. To eliminate this non-detectable yet existing leukaemic cell mass, maintenance therapy is given for an extended period (2 to 2.5 years) with the purpose of reducing the possible relapse and possibly eradicating this "iceberg" like leukaemic mass.
These residual laeukemia blasts remaining in the patient, which cannot be detected by the available techniques comprise the minimal residual disease (MRD) i.e. described as Phase B, C and D in results. It is the proliferation and infiltration of these blast cells which serves as the major cause of relapse. This "iceberg" is conventionally addressed as minimal residual disease (MRD) (Knechtli et. al. J. Clin Path 48, 1995). It is defined as the presence of leukaemic cells not detectable by morphology. Assays to detect minimal residual disease (MRD) is the need of the hour as these will help the clinicians to assess the effect of treatment on tumor burden and allow anticipation of relapse with greater precision (Brisco, Condan, Hughes, et. al. The Lancet 1994).
Therefore, the applicants have developed a simple blood based lymphoproliferative assay to detect minimal residual disease (MRD) and predict relapse in Acute lymphoblastic leukaemia (ALL) patients employing AchatininH as the tool.